Fig. 4.

BMP7 promotes proliferation of PAX2⁺ NZ cells. (A) Number of mitotic phospho-histone H3 (phospho-HH3)-positive cells are shown normalized to total number of cells in each treatment group. Values represent averages ± s.e.m. (n=3). Analysis was carried out after 16-18 hours of stimulation. ^*P=0.002 for control versus BMP7-treated samples. ^**P=0.005 for BMP7 treated versus BMP7+JNK inhibitor or BMP7+TAK1 inhibitor treated samples. (B) Number of phospho-HH3⁺ PAX2⁺ cells normalized to total cell numbers in each treatment group. Values represent averages ± s.e.m. (n=3). Analysis was carried out after 16-18 hours of stimulation. ^*P=0.03 for control versus BMP7-treated samples. ^**P=0.006 for BMP7-treated versus BMP7+JNK inhibitor-treated samples. ^***P=0.04 for BMP7-treated versus BMP7+TAK1 inhibitor-treated samples. (C) Representative images of stained NZ cells, revealing that SIX2⁺ (red) cells proliferate (phospho-HH3⁺, green) after 16-18 hours of BMP7 stimulation. DAPI stained nuclei are blue. Scale bar: 10 μm. (D) Western blot analysis of NZ cells reveals upregulation of cyclin D3 after 2 and 6 hours of BMP7 exposure. Addition of JNK inhibitor eliminates the increase in cyclin D3 in response to BMP7. β-tubulin was used as loading control. (E) Representative FACS analyses of 7AAD-stained NZ cells following 16-18 hours of stimulation, as indicated. No significant difference in the contribution of 7AAD⁺ dead cells could be detected between treatment groups. (F) Western blot analysis of cleaved caspase-3 in NZ cells following 16-18 hours of stimulation as indicated. β-tubulin was used as loading control.